TGF-xcex21 is a polypeptide growth factor that belongs to a large family of structurally related growth factors referred to as the TGF-xcex2 superfamily. It is a multifunctional cytokine that plays a major role in morphogenesis, development, tissue repair, and in the pathogenesis of fibrotic diseases (Massague, J. et al., Cancer Surv 1992;12:81-103 (1992), Norgaard, P., Hougaard, S., Poulsen, H. S. and Spang-Thomsen, M., Cancer Treat Rev 21, 367-403 (1995)). While TGF-xcex21 was originally described as an inducer of anchorage independent growth in fibroblasts, it is known to be a potent inhibitor of epithelial cell growth, though it can stimulate the growth of certain tumor cells (Huang, F., Newman, E., Theodorescu, D., Kerbel, R. S. and Friedman, E., Cell Growth Differ 6, 1635-1642 (1995), Lu, C. and Kerbel, R. S., Curr Opin Oncol 6, 212-220 (1994)). The role of TGF-xcex2 in human malignancies is complex and both paracrine and autocrine actions need to be assessed. TGF-xcex21 is elevated in several cancers including clear-cell renal carcinomas (RCCs) (Derynck, R. et al., Cancer Res 4 7, 707-712 (1987), Gomella, L. G. et al., Cancer Res 49, 6972-6975 (1989), Ramp, U. et al., J Urol 157, 2345-2350 (1997)). Some tumor cells are sensitive to TGF-xcex2""s growth suppressive effects, whereas others are not (Gomella, L. G. et al., Cancer Res 49, 6972-6975 (1989), MacKay, S. L. et al., Ann Surg 2 2 1, 767-776; discussion 776-767 (1995), Jakowlew, S. B., Mathias, A., Chung, P. and Moody, T. W., Cell Growth Differ 6, 465-476 (1995), Jakowlew, S. B., Moody, T. W. and Mariano, J. M., Anticancer Res 17, 1849-1860 (1997), and Norgaard, P., Spang-Thomsen, M. and Poulsen, H. S., Br J Cancer 73, 1037-1043 (1996)). In a recent paper, 20/20 primary RCCs and 30/30 RCC cell lines expressed TGF-xcex21, with the majority of the cell lines resistant to the growth suppressive effect of exogenous TGF-xcex21 (Ramp, U. et al, Lab Invest 76, 739-749 (1997)). Moreover, serum and urine levels of TGF-xcex21 and tissue expression of TGF-xcex21 mRNA in several cancers correlate inversely with prognosis, suggesting an important paracrine role for TGF-xcex21 in promoting tumor progression and possibly metastasis in vivo (Tsai, J. F. et al., Medicine (Baltimore) 76, 213-226 (1997), Knoefel, B. et al., J Interferon Cytokine Res 17, 95-102 (1997), Ivanovic, V., Melman, A., Davis-Joseph, B., Valcic, M. and Gellebter, J., Nat Med 1, 282-284 (1995), Friess, H. et al., Gastroenterology 105, 1846-1856 (1993), and Junker, U. et al., Cytokine 8, 794-798 (1996)).
RCC is the most common cancer of the kidney, occurring in over 27,000 individuals in the U.S. each year and is responsible for over 11,000 deaths annually (Linehan, W. M., Lerman, M. I. and Zbar, B., JAMA 273, 564-570 (1995)). The treatment of RCC remains frustrating to the oncologist; locally unresectable and metastatic disease has dismal prognosis. There is tremendous need to understand the basic biology of RCC and develop better therapeutic options. Most sporadic and hereditary RCCs (VHL-disease associated) have mutated and/or loss of both copies of the VHL gene (Linehan, W. M., Lerman, M. I. and Zbar, B., JAMA 273, 564-570 (1995)). The VHL gene product (pVHL) is lost in early atypical cysts, suggesting that pVHL might play a xe2x80x9cgatekeeperxe2x80x9d role in RCC development, analogous to the APC gene product in colon cancer (Lubensky, I. A. et al., J Pathol 149, 2089-2094 (1996) and Maher, E. R. and Kaelin, W. G., Jr., Medicine (Baltimore) 76, 381-391 (1997)). VHL-disease associated tumors are typically hypervascular and target genes identified to date include VEGF, TGF-xcex1 and PDGF-B, all of which have pro-angiogenic effects (Iliopoulos, O., Levy, A. P., Jiang, C., Kaelin, W. G., Jr. and Goldberg, M. A., Proc Natl Acad Sci USA 93, 10595-10599 (1996), Knebelmann, B., Ananth, S., Cohen, H. T. and Sukhatme, V. P., Cancer Res 58, 226-231 (1998), Mukhopadhyay, D., Knebelmann, B., Cohen, H. T., Ananth, S. and Sukhatme, V. P., Mol Cell Biol 17, 5629-5639 (1997), and Gnarra, J. R. et al., Proc Natl Acad Sci USA 93, 10589-10594 (1996)). TGF-xcex2 is another gene significantly involved in angiogenesis (Pepper, M. S., Mandriota, S. J., Vassalli, J. D., Orci, L. and Montesano, R., Curr Top Microbiol Immunol 213, 31-67 (1996)). Although TGF-xcex21 has been found to be elevated in RCCs (Derynck, R. et al., Cancer Res 47, 707-712 (1987), Gomella, L. G. et al., Cancer Res 49, 6972-6975 (1989), Ramp, U. et al., J Urol 157, 2345-2350 (1997), Ramp, U. et al., Lab Invest 76, 739-749 (1997), and Knoefel, B. et al., J Interferon Cytokine Res 17, 95-102 (1997)) there has been no link to date with the VHL tumor suppressor and no functional role has been ascribed to TGF-xcex21 for RCC growth in vivo.
The present invention is based on the discovery that TGF-xcex21 is a novel target gene for pVHL (von Hippel-Lindau) and that pVHL regulates the TGF-xcex21 gene at the post-transcriptional level. Furthermore, as described herein, evidence is presented that antagonizing the effects of TGF-xcex21 suppresses tumor growth in vivo through an anti-angiogenic mechanism.
In particular, the present invention relates to methods of inhibiting proliferative diseases in a vertebrate. The proliferative disease is characterized as having increased production of TGF-xcex2 and is further characterized by having angiogenic activity. The increased TGF-xcex2 production can be global, or be very localized, and the TGF-xcex2 can be secreted either by proliferating cells, or by stromal cells, such as fibroblasts, macrophages, platelets, endothelial calls, granular neutrophils, and other cells. Because TGF-xcex2 can autoregulate itself, and induce production of more TGF-xcex2, the cells surrounding those producing TGF-xcex2 can in turn produce TGF-xcex2. More particularly, the angiogenic activity is TGF-xcex2-mediated angiogenic activity and the inhibition of the proliferative disease results from the inhibition of TGF-xcex2-mediated angiogenesis, specifically the xe2x80x9cresolutionxe2x80x9d phase of TGF-xcex2-mediated angiogenesis. As defined herein, the xe2x80x9cresolutionxe2x80x9d phase of angiogenesis is the phase of angiogenesis where endothelial cells stop proliferating and migrating, in which basement membrane reforms and in which pericytes, the differentiation of which is known to be TGF-xcex2 dependent, attach to the basement membrane.
Although any proliferative disease characterized as above is encompassed by the present invention, in one embodiment of the present invention, the proliferative disease is cancer, or tumor growth, and the inhibition of the disease results in inhibiting the growth of a tumor, or in the regression of an already established tumor. In particular, the cancer is clear-cell renal carcinoma, or RCC.
As described herein, TGF-xcex2-mediated angiogenesis can be inhibited by contacting the proliferating cells with a molecule that inhibits (or neutralizes or antagonizes) angiogenic activity mediated by TGF-xcex2, including TGF-xcex21, TGF-xcex22, and TGF-xcex23. For example, the molecule can be an anti-TGF-xcex2 antibody, a TGF-xcex2 antagonist such as decorin or LAP, a soluble form of a TGF-xcex2 receptor or an anti-sense oligonucleotide that binds to (hybridizes with) DNA or RNA encoding TGF-xcex2, or molecules that block TGF-xcex2""s interaction with receptor(s), molecules working intracellularly, i.e., downstream, of TGF-xcex2 receptor(s).
In another embodiment of the present invention, the proliferative disease is inhibited by contacting the proliferating cells with a molecule that inhibits TGF-xcex2 activity as described above in combination with one, or more additional anti-angiogenic molecules, e.g., angiostatin or endostatin or restin, or biologically active fragments thereof. In yet another embodiment of the present invention, the proliferating cells are contacted with a chemotherapeutic, immunotherapeutic or radiologic agent in combination with the molecule that inhibits TGF-xcex2 angiogenic activity. Additionally encompassed by the present invention is any combination therapy including of molecules that inhibit TGF-xcex2-mediated angiogenic activity, other anti-angiogenic molecules, chemotherapy, immunotherapy and/or radiation therapy.
Also encompassed by the present invention is a method of monitoring tumor growth or cancer progression in a vertebrate. In one embodiment of the present invention, tumor metastases in a mammal is monitored by evaluating (e.g., determining) TGF-xcex2 levels in a biological sample obtained from the mammal considered to be at risk for tumor metastases.
In another embodiment of the present invention, the efficacy of an anti-tumor therapy is evaluated where cells obtained from the tumor are evaluated for the expression of functional TGF-xcex2 receptors. As defined herein, a functional (e.g., biologically active) TGF-xcex2 receptor is a receptor that binds TGF-xcex2, or a fragment thereof. If the proliferating cells express functional TGF-xcex2 receptors, then the anti-tumor therapy can include contacting the tumor cells with TGF-xcex2, or a biologically active fragment thereof. As defined herein, the biological activity of TGF-xcex2 includes any of the known cytokine activities of TGF-xcex2, including the ability to bind to its cognate receptor, inhibit epithelial cell growth and/or differentiation, as well as the ability to specifically bind to anti-TGF-xcex2 antibodies or to stimulate immunogenic response (e.g., elicit antibodies) in a mammal. If the proliferating cells do not express functional TGF-xcex2 receptors, or do not express TGF-xcex2 receptors at all, then the choice of therapy would include contacting the tumor cells with a molecule that inhibits TGF-xcex2-mediated angiogenic activity.
Thus, as described herein, for the first time, TGF-xcex2 is shown to be a target for the VHL (von Hippel-Lindau) tumor suppressor and a critical growth factor for clear-cell renal carcinoma. Additionally, as described herein, antagonizing TGF-xcex2 activity (in particular its paracrine activity) provides novel methods of inhibiting proliferating cells that secrete TGF-xcex2, or by inhibiting proliferating cells that cause the secretion of TGF-xcex2 by stromal cells, and more specifically, by TGF-xcex2-secreting tumors such as clear-cell renal carcinoma.